Wnt activation disturbs cell competition and causes diffuse invasion of transformed cells through NF-κB-MMP21 pathway

Normal epithelial cells exert their competitive advantage over RasV12-transformed cells and eliminate them into the apical lumen via cell competition. However, the internal or external factors that compromise cell competition and provoke carcinogenesis remain elusive. In this study, we examine the effect of sequential accumulation of gene mutations, mimicking multi-sequential carcinogenesis on RasV12-induced cell competition in intestinal epithelial tissues. Consequently, we find that the directionality of RasV12-cell extrusion in Wnt-activated epithelia is reversed, and transformed cells are delaminated into the basal lamina via non-cell autonomous MMP21 upregulation. Subsequently, diffusively infiltrating, transformed cells develop into highly invasive carcinomas. The elevated production of MMP21 is elicited partly through NF-κB signaling, blockage of which restores apical elimination of RasV12 cells. We further demonstrate that the NF-κB-MMP21 axis is significantly bolstered in early colorectal carcinoma in humans. Collectively, this study shows that cells with high mutational burdens exploit cell competition for their benefit by behaving as unfit cells, endowing them with an invasion advantage.

RasV12-transformed cells residing between normal cells are apically extruded via cell competition and are excreted along with other body waste 17 .This model serves as a valuable platform for studying the apical elimination of transformed cells in vivo and is well-suited for functional analysis of cell competition in a physiological setting [18][19][20][21] .To date, a series of studies have reported the apical extrusion of RasV12 in several mouse organs, including small intestine 17 , stomach 20 , pancreas 19 , and lung 18 .These findings indicate that the removal of transformed cells through cell competition is an innate defense system orchestrated among epithelial cells, which suppresses the accumulation of mutated lineages and reduces the risk of oncogenesis.
In general, progressive accumulation of genetic aberrations is associated with the onset of carcinogenesis 22,23 .For instance, in human colorectal cancer, the transition from normal epithelial cells to malignant cells follows a stepwise series of genetic mutations, typically involving APC, Ras, and p53, in a specific order 24,25 .Yet, the precise mechanism that determines the sequence of these genetic alterations remains unidentified.Moreover, the impact of accumulated oncogenic insults on cell competition is unclear.This caused us to wonder whether genetic mutations might influence the behavior of transformed cells in a competitive environment.We reasoned that it might be possible to exploit human familial adenomatous polyposis (FAP) as a model to evaluate functional perturbation of cell competition during multi-sequential carcinogenesis.Given that mutations in the APC tumor suppressor gene are the most prevalent initiating insults in colorectal cancer 26 , we engineered a cell competition mouse model to sustain infrequent somatic activation of Ras in APC-mutated epithelia in a manner reminiscent of FAP and investigated the fate of transformed cells.In this study, we demonstrate that APC-ablation leads to a malfunction of cell competition, converting this normal homeostatic mechanism into a potential driver of tumor progression.

Aberrant Wnt activation disturbs apical extrusion and potentiates non-cell autonomous diffuse invasion of RasV12transformed cells into the basal lamina
To examine the effect of APC deficiency on the apical extrusion of RasV12-transformed cells, we utilized APC Min mice, which harbor a heterozygous loss-of-functional mutation in the APC gene 27 .Quantitative real-time PCR (q-PCR) analysis using intestinal epithelial cells of wild-type-or APC Min -derived crypt organoid cultures confirmed a significant elevation in the expression of Wnt-targeted genes in APC Min mice compared to wild-type mice (Supplementary Fig. 1a).In order to somatically activate the H-Ras protein in a small subset of intestinal cells in the context of APC ablation, APC Min mice were bred with Villin-Cre ERT2 mice and DNMT1-CAG-loxP-STOP-loxP-HRas V12 -IRES-eGFP mice (referred to as APC Min -Villin-RasV12 mice) or CAG-loxP-STOP-loxP-eGFP mice (APC Min -Villin-GFP mice).While Villin-Cre ERT2 mice recombine Creresponsive elements in both differentiated cells and Lgr5+ stem cells 28 , low-dose, tamoxifen-dependent, mosaic expression of transgenes was primarily restricted to terminally differentiated enterocytes, but not occur in Olfm4-positive stem cells, Wheat Germ Agglutinin (WGA)positive paneth and goblet cells, or DCLK1-positive tuft cells (Supplementary Fig. 1b).This selective expression pattern is likely due to the higher tamoxifen sensitivity of differentiated cells compared to stem cells.
We next examined the fate of newly emerged RasV12-transformed cells in the intestinal tracts of 6-10-weeks-old mice, 3 days after tamoxifen administration.This age group was chosen to avoid aberrant activation of Wnt signaling caused by loss of heterozygosity (LOH) of the APC locus in older mice 29 .As a result, a substantial number of RasV12-expressing cells surrounded by normal epithelial cells were apically eliminated in Villin-RasV12 mice.Interestingly, we observed a small fraction of transformed cells (1.87 ± 0.23%) undergoing basal delamination, with nuclei oriented toward the basal lamina (Fig. 1a, b).These cells were defined as basally extruded cells.Strikingly, the percentage of cells for which the above criteria were met was significantly higher in APC Min -Villin-RasV12 mice (7.67 ± 1.03%) compared with Villin-RasV12 mice, while GFP-expressing cells of APC Min -Villin-GFP mice were either apically or basally extruded no more often than their neighbors (Fig. 1a, b).Moreover, the basally extruded APC Min /RasV12-transformed cells penetrated the basement membrane and invaded the surrounding stromal tissues, where they expanded (Supplementary Fig. 2a).Importantly, these APC Min /RasV12 cells were negative for Ki-67 staining, a surrogate marker for cell proliferation, ruling out the possibility of growth-stimulated overcrowding as the cause of their delamination (Supplementary Fig. 2b).These findings collectively suggest that Wnt activation disrupts cell competition, resulting in promoted basal extrusion of RasV12-transformed cells in vivo.Furthermore, basal extrusion of APC Min /RasV12 cells occurred with a similar frequency throughout the small intestine, from duodenum to ileum (Supplementary Fig. 3a, b).Consistent with the in vivo observations, experiments using intestinal organoids also revealed basal extrusion of APC Min /RasV12 cells under the condition of low-dose tamoxifen, while that of APC Min /GFP or RasV12-single mutated cells was rarely observed (Fig. 1c, d).Three-dimensional modeling of APC Min /RasV12-transformed organoids highlighted elongating, protruding transformed cells that were basally exterminated (Fig. 1e).Importantly, when RasV12 mutants were predominantly induced in APC Min organoids using high-dose tamoxifen, the frequency of both apical and basal extrusion was drastically reduced in various cluster-forming transformed cells, in a density-dependent fashion (Fig. 1f, g).These results emphasize that the presence of surrounding APC-ablated cells is necessary for APC Min /RasV12 cells to be expelled from the epithelia, indicating that extrusion of transformed cells occurs non-cell autonomously.
To validate that the effect of APC mutation on cell competition is primarily driven by Wnt activation, we employed a tissue injury model 30 .Treatment with indomethacin resulted in significant damage to the small intestinal tissues, resulting in a gradual reduction in body weight.However, the mice began to recover approximately 3 days after the injury (Supplementary Fig. 4a).At 8 days post-injury, we observed a marked elevation in Wnt activity (Supplementary Fig. 4b).Importantly, basal extrusion of RasV12-transformed cells was significantly enhanced compared to non-injured mice, providing further evidence that Wnt activation is the underlying cause of basal delamination of transformed cells in a competitive environment (Supplementary Fig. 4c, d).

APC Min -Villin-RasV12 mice develop invasive carcinomas
We then tracked the fate of APC Min /RasV12-transformed cells as they invaded the lamina propria long after RasV12 expression.At 21 days after tamoxifen administration, both Villin-RasV12 mice and APC Min -Villin-GFP mice displayed no overt phenotypes, with GFP-expressing cells having disappeared due to the rapid turnover of intestinal epithelial cells (Fig. 2a).In marked contrast, APC Min -Villin-RasV12 mice succumbed to intestinal transformation, with GFP-positive APC Min / RasV12 cells being confined in the stroma of upper villi (Fig. 2a).Notably, no microscopically detectable architectural deformities in the form of benign adenomatous tumors (adenoma) were observed in proximity to tumors.This implies that cancer cells were generated directly from the normal mucous membrane through the diffuse infiltration of transformants.Furthermore, the stromal cell nests appeared histopathologically undifferentiated, lacking forming glandular ducts, being different from signet-ring cell carcinomas or neuroendocrine neoplasms, and most nearly resembling diffuse-type carcinomas observed in human 31,32 .By 36 days, APC Min /RasV12 cells had further invaded the submucosa and even the muscle layer (Fig. 2b).Notably, these cells invaded cohesively, as evidenced by the sustained expression of E-cadherin in the invasive cells (Fig. 2b).
These findings prompted us to thoroughly characterize the properties of APC Min /RasV12-malignant cells by evaluating the expression of several markers (Supplementary Fig. 5a).APC Min /RasV12 carcinoma cells did not show positive staining for AB-PAS.Additionally, they expressed Sox9 (an intestinal progenitor marker) at the lowest level while maintaining the CDX2 expression (an intestinal lineage marker).In contrast, typical adenoma cells produced in elderly APC Min -Villin-GFP mice displayed the opposite expression pattern, being positive for AB-PAS staining, exhibiting high levels of Sox9 expression, and reduced CDX2 expression.Immunostaining for Ki-67 demonstrated that carcinoma cells showed moderate growth stimulation, in sharp contrast to the highly proliferative status of adenoma cells.This would reflect a stress-resistant status of APC Min /RasV12 cells to survive in a harsh stromal environment.Furthermore, APC Min   /Phalloidin GFP not express synaptophysin or chromogranin, markers for enteroendocrine cells, confirming that they were not derived from enteroendocrine cells (Supplementary Fig. 5b).These results indicate that Wnt-induced perturbation of cell competition leads to the generation of highly invasive, unique carcinoma cells derived from terminally differentiated cells, and follow a distinct histopathogenesis pathway from the well-known adenoma-carcinoma sequence.Importantly, APC Min /RasV12-positive adenomatous structures, where GFP-positive transformants occupied positions ranging from crypts to differentiated tips of villi, were also observed, albeit rarely, plausibly representing stem cell-hit lesions.Of particular interest, APC Min / RasV12-transformed cells actively infiltrated stromal vessels (Fig. 2c), and three-dimensional imaging of the small intestine by whole-mount staining revealed their preferential invasion of lymphatic vessels (Fig. 2d).Moreover, APC Min -Villin-RasV12 mice with lymphatic invasion invariably exhibited metastasis of tumor cells to mesenteric lymph nodes (Fig. 2e).In summary, dysregulation of cell competition caused by Wnt activation can be exploited by RasV12-transformed differentiated cells to manifest diffuse invasion, resulting in the production of highly invasive carcinoma cells, which follows a distinct fate from the adenoma-carcinoma sequence.

MMP21 regulates non-cell autonomous basal extrusion of APC Min /RasV12 cells
With the aim of understanding the molecular mechanism underlying the non-cell autonomous basal delamination of transformed cells, we established Madin-Darby canine kidney (MDCK) or MDCK-pTR GFP-RasV12 cells (tetracycline-inducible RasV12-expressing cells) stably expressing a mCherry-conjugated β-catenin mutant (β-cat ΔN) to mimic Wnt activation in vitro (Fig. 3a).This mutant lacks the N-terminal 131 amino acids and fails to undergo GSK3β-mediated degradation, thereby resulting in constitutive activation of Wnt signaling 33 .We confirmed that MDCK cells expressing a β-cat ΔN mutant (referred to as β-cat ΔN cells) and MDCK-pTR GFP-RasV12 cells expressing the same mutant (referred to as β-cat ΔN/RasV12 cells) exhibit comparable Wnt activation by TOP FLASH reporter assay (Fig. 3b).When β-cat ΔN/ RasV12-transformed cells were co-cultured with β-cat ΔN cells at a ratio of 1:50, a sizeable fraction of β-cat ΔN/RasV12 cells underwent basal extrusion into the collagen matrix over time, while the number of apically extruded cells or dead cells slightly increased compared to βcat ΔN/RasV12 cells cultured alone (Fig. 3c, d).In addition, we found that β-cat ΔN/RasV12-transformed cells surrounded by themselves remained within epithelia, indicating that the presence of surrounding β-cat ΔN cells greatly accelerated basal extrusion of β-cat ΔN/RasV12 cells.Thus, we successfully phenocopied the salient feature of cell density-dependent basal extrusion of APC Min /RasV12 cells in vitro.
We next wondered whether Wnt-activated cells would be eliminated by cell competition upon the emergence of Wnt-transformed cells within normal epithelia.To address this question, we established MDCK cells stably expressing doxycycline-inducible β-cat ΔN cells (Supplementary Fig. 6a).After 16 h of doxycycline treatment, Wnt signaling was profoundly activated (Supplementary Fig. 6b).However, we did not observe any apparent "loser" phenotypes such as cell extrusion or cell death when β-cat ΔN cells were surrounded by normal MDCK cells (Supplementary Fig. 6c, d).This indicates that Wnt activation alone does not confer loser status, at least under these experimental conditions.It is worth noting that in other systems, such as zebrafish or hair follicles of mice, cells with activated Wnt signaling have been reported to be expelled from tissues via cell competition 34,35 , suggesting that the removal of Wnt mutants by cell competition may be context-dependent.
To uncover the underlying molecular mechanism whereby Wnt activation potentiates basal extrusion of β-cat ΔN/RasV12-transformed cells, we conducted microarray analyses to search for differentially expressed genes in β-cat ΔN/RasV12 cells when co-cultured with β-cat ΔN cells (the data were deposited into the Gene Expression Omnibus (GEO) under accession number GSE217830).Among the top upregulated genes, we identified MMP21, a member of the matrix metalloproteases (MMPs) superfamily (Fig. 3e, f).MMPs play crucial roles in tumor progression by degrading and remodeling extracellular matrix (ECM), facilitating the invasion of malignant cells into stomal tissues 36 .This led us to assess expression levels of other MMP molecules by q-PCR analysis, which revealed that MMP21 was by far the most abundant MMP expressed in β-cat ΔN/RasV12 cells co-cultured with βcat ΔN cells (Fig. 3g).These findings suggest that MMP21 may have a unique function among MMPs and could represent a particular target for the treatment of diffuse-type carcinomas.Immunofluorescence analysis, however, did not show overt alteration in endogenous MMP21 expression.MMP21 is synthesized as a 62-kDa proprotein, which is then activated through cleavage of the prodomain by furin or related proteases, resulting in the secretion of the active 49-kDa protease into the extracellular milieu 37 .For this reason, we surmised that synthesized MMP21 is rapidly secreted out of cells, making it difficult to observe intracellular MMP21.To circumvent this difficulty, we hampered the transport machinery by treating cells with brefeldin A (BFA).This manipulation led to the accumulation of MMP21 in β-cat ΔN/RasV12 cells when surrounded by β-cat ΔN cells, whereas MMP21 levels in β-cat ΔN/RasV12 cells cultured alone in the presence of BFA elevated to a lesser extent (Fig. 3h, i).Human MMP21 is the most recently cloned MMP gene 38 , and its physiological function, including substrate specificity, is not fully understood.Based on its amino acid sequence, MMP21 cannot be classified as a collagenase, gelatinase, stromelysin, matrilysin, or membrane-type MMP 38 .To set out to characterize the proteolytic capabilities of MMP21, we produced a recombinant protein of the catalytic domain of MMP21 using E. coli since the latent MMP21 protein is catalytically inert (Supplementary Fig. 7a).This recombinant protein was incubated with various ECMs, which are principal constituents of connective tissues.The catalytic domain of MMP21 extensively degraded collagen type IV and digested collagen type I and fibronectin, generating both small and large fragments.In contrast, laminin was resistant to hydrolysis, suggesting that MMP21 is a competent Zn 2+ -dependent endoproteinase with unique specificity for Fig. 1 | APC Min /RasV12-transformed cells are basally delaminated in a non-cell autonomous fashion.a, c, e, f Confocal microscopic images of intestinal villi (a) or intestinal organoids (c, e, f) from APC Min -Villin-GFP, Villin-RasV12, or APC Min -Villin-RasV12 mice.In a, APC Min -Villin-GFP, Villin-RasV12, or APC Min -Villin-RasV12 mice were injected with low-dose tamoxifen and were sacrificed 3 days later.Frozen sections were stained with DAPI (blue), anti-GFP antibody (green), and phalloidin (red).In c, e, f, intestinal organoids from APC Min -Villin-GFP, Villin-RasV12, or APC Min -Villin-RasV12 mice were treated with 100 nM (c, e) or 1 μM tamoxifen (f) and cultured for the indicated times (d) or 24 h (e, f).Ba and Ap stand for the basal and apical sides, respectively (a, c, f).White squares in the left images are magnified in the corresponding right panels (a, c, f).White arrowheads depict the apically extruded cells whereas white arrows indicate the basally extruded cells, respectively (a, c, e).In (f), An arrow indicates a single transformed cell that is basally extruded (left magnified image) while the clustered transformed cells that are not extruded are depicted by an arrowhead (right magnified image).Scale bars, 50 μm (a, c), 100 μm (e), and 10 μm (f).b, d, g Quantification of apical and basal extrusion of transformed cells in small intestine (b) or intestinal organoids (d, g).'Not extruded' indicates cells remaining within epithelium.'Apical extruding' indicates apically extruding cells, with their nucleus apically shifted, that are still attached to the basement membrane.'Apical extruded' indicates cells completely detached from the basement membrane and 'Basal extrusion' indicates cells delaminating into the basal lamina.Data are mean ± s.e.m. n = three independent experiments.*P = 0.0054 for basally extruded cells between RasV12-and APC Min /RasV12-expressing cells (b), unpaired two-tailed t-test.In g, the clusters are categorized by the number of APC Min /RasV12 cells present in each cluster.Data are mean ± s.e.m. n = three independent experiments.*P = 0.0037; **P = 0.0007 for cells with extrusion, unpaired two-tailed t-test.
certain substrates (Supplementary Fig. 7b).To examine the functional involvement of MMP21 in basal extrusion of transformants, we generated β-cat ΔN/RasV12 cells stably expressing MMP21 short-hairpin RNA (shRNA) (Fig. 3j).MMP21-knockdown profoundly prevented the basal extrusion of β-cat ΔN/RasV12 cells (Fig. 3k, l), highlighting an active role of MMP21 in the non-cell autonomous basal extrusion of transformed cells.To bolster evidence for the requirement of MMP21 in this process, we investigated the effect of a Pan-MMP inhibitor, GM6001, and found that the frequency of basal extrusion was compromised to the same extent as MMP21-knockdown (Supplementary Fig. 7c, d).
We further investigated the functional relevance of MMP21 in the basal invasion of APC Min /RasV12 cells in vivo.To begin with, we examined MMP21 expression in intestinal epithelia and observed profound upregulation of MMP21 in RasV12-expressing cells that were produced mosaically within the APC Min -mutated epithelia, but not in GFP-expressing cells nor in single RasV12-transformed cells (Fig. 4a).
To investigate the cell density-dependent effect on MMP21 expression, intestinal organoids derived from APC Min -Villin-RasV12 mice were treated with either low-dose or high-dose tamoxifen.Mosaic expression of RasV12 mutants under the APC Min -background induced elevated expression of MMP21, but not in APC Min /RasV12 cells surrounded by one another, nor in APC Min /GFP cells or RasV12 cells (Fig. 4b), recapitulating the cell density-dependent upregulation of MMP21 in APC Min /RasV12 cells.It is worth noting that MMP21 was upregulated not only in basally extruded cells but also in cells remaining within the epithelia or even apically extruding cells.This suggests that the elevated expression of MMP21 alone is not sufficient to redirect transformed cells to undergo basal delamination, but rather it promotes this process as one of the priming factors.To examine the functional  significance of MMP21 in basal delamination from normal mucosa, we utilized MMP21-deficient mice (Fig. 4c-f).Although the majority of the progeny (93.9%; n = 132 mice) died immediately after birth, likely due to congenital heart disease caused by MMP21 abrogation 39,40 , we analyzed the APC Min -Villin-RasV12-MMP21 KO mice that were born healthy, grew normally, and did not exhibit apparent diseases.Consequently, MMP21-deficient APC Min /RasV12 cells were incompetent to drive the basal extrusion of transformed cells (Fig. 4g, h).These results collectively demonstrate that MMP21 is one of the molecules that govern the diffuse invasion of transformed cells.

NF-κB signaling directly elevates MMP21 expression
To identify the upstream regulator(s) responsible for the upregulation of MMP21 expression, we initially examined the involvement of the Notch and TGF-β pathways as potential regulators of MMP21 41,42 .However, no alterations in activities of these pathways were evident by examining the transcriptional signature of β-cat ΔN/RasV12 cells cocultured with β-cat ΔN cells, armed with the microarray datasets (Fig. 3e).Subsequently, we conducted gene-set enrichment analyses (GSEA) to gain a comprehensive overview of differentially expressed genes, and found that the production of IL-6 and chemokines substantially increased (Fig. 5a).This result is suggestive of activation of the NF-κB signaling pathway 43 , we thereby conducted a reporter assay using a plasmid with luciferase expression under the control of tandem repeats of the NF-κB transcriptional response element.Consequently, we validated the non-cell-autonomous activation of NF-κB signaling (Fig. 5b).Notably, we observed negligible activation of Wnt signaling (Supplementary Fig. 8), indicating that further enhancement of Wnt signaling does not occur in β-cat ΔN/RasV12 cells.To explore whether the upregulation of MMP21 and NF-κB activation induced in β-cat ΔN/ RasV12 cells are specific to a Wnt-activated background, we performed microarray analysis to profile RasV12-single transformed cells cocultured with normal cells, alongside RasV12 cells cultured alone (GEO accession Number: GSE236658).As a result, we did not observe a significant elevation of MMP21, and NF-κB activation was found to be negligible.These findings suggest that a Wnt-activated environment is necessary for these alterations to occur.To investigate the functional relevance of the NF-κB pathway in MMP21-mediated diffuse invasion of transformed cells, we treated the cells with BAY 11-7082, an inhibitor of NF-κB that targets IκB kinase (IKK).Consequently, BAY 11-7082 substantially diminished the cell density-dependent upregulation of MMP21, suggesting that NF-κB signaling acts as an upstream regulator of MMP21 expression (Fig. 5c, d).We next queried whether the NF-κB complex directly regulates MMP21 transcription.To this end, we performed chromatin immunoprecipitation (ChIP) assay followed by q-PCR using primers encompassing the promoter region of the MMP21 locus, which contains the predicted p65 binding sequence according to the JASPAR2014 program (Fig. 5e).The ChIP q-PCR experiments demonstrated that activation of NF-κB signaling by TNF-α treatment significantly increased the association of p65 with the promoter region of MMP21, indicating direct regulation of MMP21 expression by the NF-κB signaling pathway (Fig. 5e).Furthermore, inhibition of NF-κB signaling profoundly suppressed the basal extrusion of β-cat ΔN/RasV12 cells co-cultured with β-cat ΔN cells, and adversely enhanced their apical elimination in a dose-dependent manner (Fig. 5f, g).These results indicate that NF-κB signaling is crucial in determining the direction of cell extrusion, and its activation redirects transformed cells toward basal delamination, with MMP21 being one of the downstream targets.
To evaluate the activity of NF-κB signaling in vivo, we examined the expression of p65 and observed a profound accumulation of nuclear p65 in APC Min /RasV12 cells within the intestinal epithelia, which was otherwise unaffected in APC Min /GFP-or single RasV12-mutated cells (Fig. 5h).Furthermore, p65 elevation occurs in a cell densitydependent fashion, as observed in intestinal organoids where APC Min / RasV12 cells surrounded by APC Min cells exhibited increased nuclear p65 levels whereas p65 intensity in APC Min /RasV12 mutants surrounded by themselves was no higher than that in RasV12 or APC Min /GFP cells (Fig. 5i).To investigate the impact of NF-κB signaling inhibition on MMP21 expression and the basal extrusion of APC Min /RasV12 cells, we treated intestinal organoids with BAY 11-7082.The treatment drastically diminished the cell density-dependent upregulation of MMP21 and counteracted the basal extrusion of APC Min /RasV12 cells (Fig. 5j-l).Additionally, we challenged APC Min -Villin-RasV12 mice with SN50, a cellpermeable NF-κB inhibitory peptide, and observed a significant decrease in the number of basally extruded cells compared to the group administered with PBS (Fig. 5m, n).These in vivo findings support our conclusion that NF-κB signaling positively regulates basal invasion of transformed cells through MMP21 upregulation.
To elucidate the mechanisms underlying NF-κB activation, we reanalyzed the microarray data, where the expression profile of β-cat ΔN/ RasV12 cells co-cultured with β-cat ΔN cells was compared to that of βcat ΔN/RasV12 cells cultured alone (Fig. 3e).Metascape analysis revealed a marked enhancement of the innate immune response including interferon production (Supplementary Fig. 9a).Notably, the retinoic acid-inducible gene-I (RIG-I) pathway emerged as one of the top-activated pathways.Additionally, Toll-like Receptor (TLR) signaling was found to be activated, with TLR3 showing the highest upregulation among the TLR family.This led us to further investigate the expression of RIG-I (also referred to as DDX58) and TLR3.Accordingly, we performed q-PCR analysis for each transcript and observed accelerated expression of both molecules in β-cat ΔN/RasV12 cells co-  9b).As RIG-I and TLR3 are well-known pattern recognition receptors to sense intracellular DNA/RNA and potentiate the innate immune response through NF-κB pathway 44,45 , we examined the inhibitory effect of these pathways on the cell fates of transformed cells.As a result, inhibition of RIG-I or TLR3 by RIG012 or CU-CPT 4a, respectively, significantly suppressed non-cell autonomous NF-κB activation and MMP21 upregulation (Supplementary Fig. 9c-e).Moreover, the frequency of basal extrusion was reduced, while apical extrusion was adversely increased, recapitulating the effect of NF-κB inhibition (Supplementary Fig. 9f, g (Supplementary Fig. 9h-j).Based on these findings, it is suggested that the competitive environment entails the RIG-I/TLR3-mediated innate immune response for Wnt-activated RasV12-transformed cells, priming them for basal infiltration through the NF-κB-MMP21 pathway (Supplementary Fig. 10).
The NF-κB-MMP21 pathway is activated in early colorectal cancers in humans Finally, we investigated whether the molecular mechanism underlying the onset of invasive carcinomas developed in APC Min -Villin-RasV12 mice is involved in human colorectal cancer.However, the cancer histopathology of APC Min -Villin-RasV12 mice does not mimic any specific pathological classes of regular human colorectal cancer, thereby, it is hard to obtain corresponding human clinical samples.Moreover, cell competition is the event occurring in the initial phase of carcinogenesis.We therefore collected 9 tissue specimens from patients diagnosed with early colorectal cancer who received endoscopic treatments.These samples were stained for MMP21, p65, β-catenin, or p-ERK.β-catenin and p-ERK were analyzed to assess the activity of Wnt signaling and MAPK signaling, respectively.Histological scores (Hscores) were calculated based on the percentages of positive cells showing, negative, weak, moderate, or strong staining intensity (Fig. 6a).Our analysis revealed higher expression of MMP21, nuclear βcatenin, p-ERK, and nuclear p65 compared to normal tissues (Fig. 6b).Furthermore, the H-score of MMP21 positively correlated with that of nuclear β-catenin, p-ERK or nuclear p65 in tumor tissues (Fig. 6c).In contrast, the H-scores of p-Akt and Cyclin E1, which are known to frequently increase in the relatively advanced stages of colorectal cancers 46,47 , did not correlate with the H-score of MMP21, despite their higher expression in tumors (Fig. 6b, c).Collectively, these results suggest that the NF-κB-MMP21 pathway is bolstered in early colorectal cancers with activated Wnt and MAPK signaling, potentially playing a role in the early infiltration of malignant cells in humans.

Discussion
There has been a surge of interest in cell competition for developing novel anti-cancer therapy.To achieve this goal, it is crucial to fully understand whether and how cell competition malfunctions during the onset of carcinogenesis.In this study, we demonstrated that preceding Wnt activation tips the balance in cell competition-induced cellular extrusion, which leads to adverse outcomes, illuminating an unanticipated outcome of cell competition.
Regarding the molecular mechanisms underlying APC Min -induced functional perturbation of cell competition, we carefully investigated potential cellular aberrations that could be attributed to the disoriented extrusion of RasV12-transformed cells.Previous studies have reported that spindle misorientation is associated with irregular extrusion in APC-mutated dividing crypt cells [48][49][50] .However, basally extruding APC Min /RasV12 cells observed in our study were terminally differentiated and non-proliferative enterocytes, suggesting that abnormal cell division is not responsible for basal extrusion.Also, APC is involved in the regulation of cellular polarity 51,52 .This led us to examine the localization of junctional molecules, but we did not observe any disturbances in either APC Min /RasV12 cells or β-cat ΔN/ RasV12 cells.From a molecular standpoint, our findings indicate that activation of RIG-I and TLR3 signaling pathways potentiates NF-κB signaling in APC Min /RasV12 cells surrounded by APC Min cells.This leads to elevated MMP21 expression, which is crucial for basal delamination.Moreover, we discovered that NF-κB signaling acts as a key determinant to switch the predominant direction of extrusion, shifting it from apical to basal.These findings align with previous studies demonstrating the role of NF-κB signaling in chronic inflammation, where it serves as a master regulator connecting inflammation and cancer, promoting cancer cell invasion 53 .While MMP21 abrogation did not fully recapitulate the phenotype observed with NF-κB inhibition, it suggests that MMP21 acts as a downstream effector of NF-κB signaling in driving diffuse invasion.Further investigation is needed to clarify how the molecular landscape is remodeled to alter the cell fates of APC Min /RasV12 compound mutants, which could be harnessed for therapeutic applications.
MMPs comprise a family of topologically related zinc endoproteases that degrade components of the extracellular matrix, or cause the shedding of membrane-anchored proteins and are involved in cancer, arthritis, multiple sclerosis, and cardiovascular disease 54 .Despite sharing a conserved catalytic domain containing a zincbinding motif (HEXXHXXGXXH), substrate specificity differs widely.In this study, we demonstrated that MMP21 exerts catalytic activity against collagen type I, type IV, and fibronectin.Although the upstream regulators for MMP21 expression were unclarified, our findings indicate that NF-κB signaling directly regulates the transcription of MMP21.However, the fact that inhibition of NF-κB signaling does not completely abolish the non-cell autonomous upregulation of MMP21 and expression of several MMPs is also under the control of NF-κB activity suggests the involvement of additional factors beyond NF-κB signaling in the regulation of MMP21 expression 55 .Previous studies have shown that increased expression of MMP21 is associated with poor clinical outcomes in several types of cancers 56 .Additionally, in this study, we observed significant upregulation of MMP21 in early human colorectal cancers, and its expression positively correlated with NF-κB signaling, Wnt signaling, and MAPK pathways.Thus, the present study is indicative of MMP21 as a potential therapeutic target for suppressing the expansion of early-phase tumors.
The adenoma-carcinoma sequence model for colorectal cancer is broadly acknowledged 57 .Nevertheless, our mouse model delineates a distinct mode of tumourigenesis, where malignant cells are diffusively generated within otherwise histologically normal tissues without the presence of adenoma components in proximity to tumors.What causes this difference in adenoma-carcinoma cancer or the histopathogenesis of APC Min -Villin-RasV12 mice is of particular interest.One possible explanation is the different origins of cancer cells.In this study, we engineered mice to induce RasV12 mutation in terminally differentiated enterocytes.However, it is well known that cancer arises from an incipient transformation event occurring primarily, but not exclusively, in the stem cell compartment.This is because stem cells in various organs are intrinsically vulnerable to genetic mutations due to their rapid cell division 58,59 .Hence, the current paradigm suggests that cancer arises from stem cells bearing sporadic mutation(s) that are inherited by all their progeny 60 .This renders a clonal advantage to transformed cells, leading to the development of adenomatous transition, followed by incidental production of invasive cells in later stages.Interestingly, several recent studies have reported that APCmutated crypt clones exhibit a competitive advantage over the surrounding normal crypts, causing accelerated expansion of APCmutated clones [61][62][63] .This suggests that APC-mutated crypts, which contain intestinal stem cells, act as winners in "crypt competition".Although studies of human colorectal cancer originally put forward stem cells as the origin of the disease, several models have demonstrated that differentiated cells can also become principal contributors to tumor development [64][65][66][67] .Thus, it is tempting to speculate that differentiated cells with deleterious mutations cause different cancerous phenotypes, such as diffuse-type cancer, albeit rarely, due to the low incidence of somatic mutations in these cells.Supporting this notion, it has been estimated that patients with de novo diffuse-type colorectal cancer are quite rare or underestimated since it is barely detectable microscopically 68 .Furthermore, diffuse-type cancers generated in the digestive tract are difficult to treat, due to scarcely noticeable early symptoms and highly invasive characteristics of cancer cells.Therefore, it is of clinical significance to delineate the nature of the disease's pathogenesis, and there is a need for sensitive, novel treatments.Whether the NF-κB-MMP21 pathway is generally relevant to other diffuse-type cancers demands future investigation.
In conclusion, our findings demonstrate that Wnt activation disrupts cell competition, and confers invasive properties on transformed cells to escape primary epithelial sites.Previous studies have also shown that the fate of transformed cells upon cell competition can differ, depending on the preceding mutation background 69,70 .This study further brings forth the prospect that cell competition constrains the order of appearance of mutations during tumor development, highlighting a link between cell competition and carcinogenesis.

Immunostaining and western blotting
For immunofluorescence, MDCK-pTR GFP-RasV12 mCherry-β-catenin Δ131, MDCK-pTR GFP-RasV12 mCherry-β-catenin Δ131 MMP21 shRNAs, MDCK-pTRE3G mCherry-β-catenin Δ131 cells were mixed with MDCK or MDCK mCherry-β-catenin Δ131 cells at a ratio of 1:50 and plated onto collagen-coated coverslips 73 .The mixture of cells was incubated for 12-24 h, followed by tetracycline or doxycycline treatment for 24 h except for analyses of basal extrusions, which were examined after 48 h of tetracycline addition.Cells were fixed with 4% paraformaldehyde (PFA) in PBS and permeabilized with 0.5% TritonX-100/PBS 74 .Primary antibodies were used at 1:100 or 1:200, and all secondary antibodies were used at 1:200.Alexa-Fluor-647-conjugated phalloidin was incubated for 1 h at an ambient temperature.For immunofluorescence using intestinal organoids, cells grown in Matrigel were incubated with Cell Recovery Solution (Corning) for 4 min before fixation with 4% PFA.After fixation, cells were permeabilized in 0.5% Triton X-100/PBS for 1 h and blocked in 1% BSA/PBS for 1 h.For immunohistochemical examinations of the small intestine, tissues were isolated, fixed with 10% formalin solution for 24 h, and embedded in paraffin or Tissue-Tek O.C.T. compound (Sakura Finetek Japan Co., Ltd.).For paraffin-embedded samples, the continuous 5 μm-thick sections were sliced.The antigen retrieval was carried out by heating slides for 40 min in 10 mM citrate (pH 6.0) solution.To carry out immunofluorescent staining, the sections were blocked with Block-Ace (DS Pharma Biomedical) and permeabilized with 0.1% Triton X-100 in PBS.For DAB staining, after primary and secondary antibody reactions, the samples were developed with DAB (Nichirei Biosciences Inc.) for 10 min, and counterstained with hematoxylin (Sakura Finetek Japan Co., Ltd.) or methyl green (FUJIFILM Wako).Subsequently, sections were dehydrated with alcohol gradient and treated with xylene to render the slice transparent.To conduct HE staining, paraffinembedded samples were sliced, deparaffinized, and stained with hematoxylin for 3 min and stained with eosin solution (Sakura Finetek Japan Co., Ltd.) for 30 sec.For frozen samples, 10 μm-thick sections were cut on a cryostat.The sections were blocked with Block-Ace and permeabilized with 0.1% Triton X-100 in PBS.Primary or secondary antibodies were incubated overnight at 4 °C or 4 h at ambient temperature, respectively.All primary antibodies were used at 1:200, and secondary antibodies were at 1:500.Whole-mount immunostaining of mouse small intestinal villi was performed as previously described 75 .Briefly, the small intestine of mice was cut out and put longitudinally on dishes to expose the lumen.After several washes with PBS, tissues were pinned on silicon plates and then fixed with 10% Formalin Solution overnight at 4 °C.The samples were then washed with PBS several times and subsequently dehydrated with 10% sucrose in PBS for 2 h, followed by incubation with 20% sucrose and 10% glycerol in PBS overnight at 4 °C afterward.After blocking with 3% donkey serum (Jackson Immunoresearch Laboratories) in 0.5% Triton-X 100 in PBS for 2 h, samples were incubated with the indicated primary antibodies diluted in the blocking solution overnight at 4 °C, followed by secondary antibodies reaction.After washing with PBS, samples were mounted with Mowiol (Calbiochem).Immunofluorescence images of intestinal tissues and cultured cells were acquired using the Olympus FV1000 system, Nikon A1R system, or KEYENCE BZ-x800.Images were quantified using the MetaMorph software (Molecular Devices) or the ImageJ/Fiji software.For western blotting, primary antibodies were used at 1:1000 17 .The western blotting data were analyzed using Ima-geQuant LAS-3000 (GE Healthcare).Uncropped scans of all blots are shown in the Source Data file.

Reverse transcription and quantitative real-time PCR
MDCK-pTR GFP-RasV12 mCherry-β-catenin Δ131 cells or a 10:1 mix culture of MDCK mCherry-β-catenin Δ131 and MDCK-pTR GFP-RasV12 mCherry-β-catenin Δ131 cells were cultured at a density of 1.2 × 10 7 cells on collagen-coated 10 cm dishes (Corning).After incubation with tetracycline for 24 h, GFP-positive β-cat/RasV12 cells were separated with an analytical flow cytometer.Total RNA was extracted from the isolated cells using ISOGENII and reverse transcribed using a PrimeScript II Reverse Transcriptase (TAKARA) and Oligo(dT)15 Primer (TAKARA), Random Primer 80 nmol (TAKARA), Deoxynucleotide Mix, 10 mM Molecular Biology Reagent (Sigma), RNase Inhibitor, Recombinant (Toyobo Life Science).Luna Universal qPCR Master Mix (New England BioLabs) was used to perform q-PCR using Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems) or QuantStudio 1 Real-Time PCR system (Applied Biosystems).The same procedures were conducted to evaluate Wnttargeted genes using intestinal organoids or epithelial cells collected from the small intestine of mice.We used β-actin as a reference gene to normalize data.The primer pairs used in the above analyses are listed in Supplementary Tables 1 and 2.

Patient samples
Early colorectal tumor specimens were provided by the Department of Pathology at the University of Tokyo Hospital.We collected 9 formalin-fixed paraffin-embedded clinical samples from patients who were diagnosed with early colorectal cancer and underwent endoscopic treatments by endoscopic mucosal resection (EMR) or endoscopic submucosal dissection (ESD) (sex, male: n = 6, female:n = 3; age, 70-82 (median = 76)).Clinical data were collected from electronic medical records.All procedures were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and later versions.The institutional Review Board of Tokyo University Hospital approved the use of the abovementioned clinical samples (no.0542-( 7)) for this study, and written informed consent was obtained from all patients.

Reporter assay
To monitor Wnt activity, MDCK, MDCK mCherry-β-catenin Δ131, MDCK-pTR GFP-RasV12, MDCK-pTR GFP-RasV12 mCherry-β-catenin Δ131 or MDCK-pTRE3G mCherry-β-catenin Δ131 cells were co-transfected with TOP FLASH or FOP FLASH and pRL-TK (Renilla luciferase plasmid).After 24 h, cells were trypsinised, and cultured alone or co-cultured with the indicated cells for 8 h, followed by tetracycline or doxycycline treatment for 16 h.Firefly luciferase activity was measured using Dual-Luciferase Reporter assay (Promega) and normalized by Renilla luciferase activity using SpectraMax iD5 (Molecular Devices).To evaluate the activity of NF-κB signaling, MDCK-pTR GFP-RasV12 mCherry-βcatenin Δ131 cells were transfected with a pGL4.32 reporter plasmid encoding for the firefly luciferase gene under the control of promoters containing tandem NF-κB elements (Promega) along with a pRL-TK vector.Subsequently, cells were cultured alone or co-cultured with MDCK mCherry-β-catenin Δ131 cells in the absence or presence of RIG012 or CU-CPT 4a and subjected to the same procedure above.

ChIP assay
Chromatin immunoprecipitation (ChIP) was performed using the SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads, Cell Signaling Technology, 9005S) following the manufacturer's instructions.Briefly, MDCK-pTR GFP-RasV12 mCherry-β-cateninΔ131 cells were treated with or without 10 ng ml -1 TNF-α for 2 h.Subsequently, cells were cross-linked with 1% formaldehyde in PBS for 10 min at room temperature and quenched with 2.5 M glycine for 5 min.Nuclei were prepared, and chromatin was incubated with micrococcal nuclease at 37 °C for 20 min, and subjected to sonication using Sonifier 250A (BRANSON) to produce chromatin smears with an average size of 150-900 bp.The soluble chromatin supernatants (Input samples) were immunoprecipitated with the rabbit anti-p65 antibody (Cell signaling) alongside rabbit IgG control (Cell signaling) overnight at 4 °C.The immunocomplexes were then rotationally incubated with ChIP-Grade Protein G Magnetic Beads for 2 h at 4 °C.ChIP DNA was eluted in ChIP elution buffer (IP sample) and subjected to q-PCR analyses using the following primers, 5'-GGCAACCACTGGGTCTGACT-3' and 5'-TCCGTACGCTGTCAAGTA TTTGAA-3'.Cycle threshold (Ct) values were normalized to the 2% input sample (percentage of input = 2% × 2 (C[T] 2% Input sample−C[T] IP sample) ).

Fig. 3 |
Fig. 3 | MMP21 plays a crucial role in basal extrusion of β-cat ΔN/RasV12 cells surrounded by β-cat ΔN cells.a, b Establishment of MDCK mCherry-β-cateninΔ131 and MDCK-pTR GFP-RasV12 mCherry-β-cateninΔ131 cells.a Constitutive expression of mCherry-β-cateninΔ131 and tetracycline-induced expression of RasV12.b TOP FLASH reporter assay.Indicated cell lines were co-transfected with TOP FLASH or FOP FLASH and Renilla expressing vector, and subjected to measurement of luciferase activity.Data are mean ± s.e.m. *P = 0.0015; **P = 0.0032, unpaired two-tailed t-test; n = three independent experiments.c Immunofluorescence images of β-cat ΔN/RasV12 cells cultured alone or surrounded by β-cat ΔN cells.d Quantification of cell fates of β-cat ΔN/RasV12 cells cultured alone or surrounded by β-cat ΔN cells.Data are mean ± s.e.m. n = three independent experiments.*P = 0.0190 for basal extrusion between two conditions, unpaired two-tailed t-test.e Experimental design and heatmap for differentially expressed genes between β-cat ΔN/RasV12 cells cultured alone and β-cat ΔN/RasV12 cells co-cultured with β-cat ΔN cells.f Volcano plot showing the distribution of fold changes and corrected P-values calculated by unpaired two-tailed t-test in β-cat ΔN/RasV12 cells mixed with β-cat ΔN cells compared with a single culture.The red or blue dots represent upregulated or downregulated genes with statistical significance, respectively.The upregulated and downregulated genes are shown in Supplementary Data 1. g q-PCR analysis of MMP-related metalloproteases.β-cat ΔN/RasV12 cells were co-cultured with β-cat ΔN cells or cultured alone, and subjected to q-PCR analysis.Values are shown as fold change relative to β-cat ΔN/RasV12 cells cultured alone.Data are mean ± s.e.m. n = three independent experiments.*P = 0.0221, unpaired two-tailed t-test.h Immunofluorescence images of MMP21.β-cat ΔN/RasV12 cells were cultured alone or co-cultured with β-cat ΔN cells in the presence of 2.5 μg ml −1 BFA.Cells were stained with Hoechst 33342 (blue) and anti-MMP21 antibody (white).i Quantification of fluorescence intensity of MMP21.Values are expressed as a ratio relative to β-cat ΔN/RasV12 cells cultured alone.Data are mean ± s.e.m. *P < 0.001, unpaired two-tailed t-test; n = 286 and 284 cells pooled from three independent experiments.j Establishment of MDCK-pTR GFP-RasV12 mCherry-β-cateninΔ131 cells stably expressing MMP21 shRNA1 or MMP21 shRNA2.Knockdown of MMP21 was confirmed by western blotting.k Immunofluorescence images of β-cat ΔN/ RasV12 cells, β-cat ΔN/RasV12 cells expressing MMP21 shRNA1 or MMP21 shRNA2 surrounded by β-cat ΔN cells.Scale bars, 10 μm (c, h, k).l Quantification of basal extrusion.Data are mean ± s.e.m. n = three independent experiments.*P = 0.0186; **P = 0.0001, unpaired two-tailed t-test.
).In organoid cultures, treatment with RIG012 or CU-CPT 4a yielded comparable effects to those observed in vitro: suppression of NF-κB activation and MMP21 elevation, and reduction of basal extrusion

Fig. 4 |
Fig. 4 | MMP21 promotes the non-cell autonomous basal invasion of APC Min / RasV12-transformed cells in vivo.a, b The non-cell autonomous upregulation of MMP21 in APC Min /RasV12 cells.a Immunofluorescence images of MMP21 in intestinal villi.APC Min -Villin-GFP, Villin-RasV12, or APC Min -Villin-RasV12 mice were injected with tamoxifen and were sacrificed 3 days later.Paraffin-embedded sections were stained with DAPI (blue), anti-GFP antibody (green), and anti-MMP21 antibody (red).b Immunofluorescence images of MMP21 in intestinal organoids from APC Min -Villin-GFP, Villin-RasV12, or APC Min -Villin-RasV12 mice at 24 h after tamoxifen treatment.Arrowheads indicate APC Min /RasV12-transformed cells with elevated MMP21 expression (a, b).c-h Effect of MMP21-knockout on basal extrusion of APC Min / RasV12 cells.c Strategy for the CRISPR-mediated MMP21-targeted mice.Physical maps of the murine MMP21 gene locus and sgRNA sequences are shown.The red line indicates the targeted region.Protospacer adjacent motif (PAM) sequences are underlined.d Genomic sequence of MMP21 locus from F1 generation MMP21-knockout mice.Note that 5890 bp covering Exon 1-Exon 7 of the MMP21 gene is deleted.e Western blot analysis of protein lysates prepared from intestinal tissues of MMP21(+/+), (+/−), (−/−) genotypes.β-actin served as a loading control.f Immunofluorescence images of MMP21 in intestinal villi from APC Min -Villin-RasV12 or APC Min -Villin-RasV12-MMP21 KO mice.Paraffin-embedded sections were stained with DAPI (blue), anti-GFP antibody (green), and anti-MMP21 antibody (red).Arrowheads indicate APC Min /RasV12-transformed cells.g, h Frequency of the basal extrusion of APC Min /RasV12 cells.g Immunofluorescence images of intestinal villi from APC Min -Villin-RasV12 or APC Min -Villin-RasV12-MMP21 KO mice 3 days after tamoxifen injection.An arrow indicates a basally extruded cell.Ba and Ap stand for the basal and apical sides, respectively (b, f, g).Scale bars, 50 μm (a) and 10 μm (b, f, g).h Quantification of apical and basal extrusion of transformed cells.Data are mean ± s.e.m. n = three independent experiments.*P = 0.0004, unpaired two-tailed t-test.

Fig. 5 |Fig. 6 |
Fig. 5 | NF-κB signaling governs basal extrusion of transformed cells via MMP21 upregulation.a, b Enhanced NF-κB activity in β-cat ΔN/RasV12 cells co-cultured with β-cat ΔN cells.a GSEA plot showing a significant correlation between the transcriptional profile of β-cat ΔN/RasV12 cells co-cultured with β-cat ΔN cells and IL-6 or chemokine production gene signatures.The P-values were calculated based on a two-sided permutation test.b NF-κB reporter assay.β-cat ΔN/RasV12 cells co-transfected with NF-κB luciferase reporter and Renilla expressing vector and were co-cultured with β-cat ΔN cells or cultured alone.Cells were then subjected to measurement of luciferase activity.Data are mean ± s.e.m. n = three independent experiments.*P = 0.0498, unpaired two-tailed t-test.c-g Effect of BAY 11-7082 on MMP21 expression and cell fates of β-cat ΔN/RasV12 cells.c Immunofluorescence images of MMP21 in the absence or presence of BAY 11-7082.β-cat ΔN/RasV12 cells were mixed with β-cat ΔN cells in the presence of BFA and were treated with DMSO or BAY 11-7082.Cells were stained with Hoechst 33342 (blue) and anti-MMP21 antibody (white).d Quantification of fluorescence intensity of MMP21.Values are expressed as a ratio relative to DMSO treatment.Data are mean ± s.e.m. *P < 0.001, unpaired two-tailed t-test; n = 418 and 420 cells pooled from three independent experiments.e p65 ChIP assay.An illustration of the p65 binding site within dog MMP21 promoter region is shown in the upper picture.Effect of TNF-α on the recruitment of p65 NF-κB to MMP21 promoter region is shown (lower panel).Data are mean ± s.e.m. n = three independent experiments.*P = 0.0069; **P = 0.0046, unpaired two-tailed t-test.f Immunofluorescence images of β-cat ΔN/RasV12 cells surrounded by β-cat ΔN cells in the absence or presence of BAY 11-7082.g Quantification of apical and basal extrusion in the absence or presence of BAY 11-7082.Data are mean ± s.e.m. n = three independent experiments.*P = 0.0018; **P = 0.0019; ***P = 0.0176; ****P = 0.0007, unpaired two-tailed t-test.h, i Activation of NF-κB signaling in APC Min /RasV12 cells.h Immunofluorescence images of p65 in intestinal villi.APC Min -Villin-GFP, Villin-RasV12, or APC Min -Villin-RasV12 mice were injected with tamoxifen and were sacrificed 3 days later.Paraffin-embedded sections were stained with DAPI (blue), anti-GFP antibody (green) and anti-p65 antibody (red).i Immunofluorescence images of p65 in intestinal organoids from indicated mice.Arrowheads indicate APC Min /RasV12-transformed cells with accumulated p65 in the nucleus (h, i).j-n Effect of NF-κB inhibition on MMP21 expression and cell fates.(j) Immunofluorescence images of MMP21 in intestinal organoids from APC Min -Villin-RasV12 mice in the absence or presence of BAY 11-7082.k Immunofluorescence images of intestinal organoids treated with 100 nM tamoxifen and cultured for 24 h in the absence or presence of BAY 11-7082.Ba and Ap stand for basal and apical sides, respectively (i-k).l Quantification of the effect of BAY 11-7082 on apical and basal extrusion ex vivo.Data are mean ± s.e.m. n = four independent experiments.*P = 0.0097 for basally extruded cells between DMSOand BAY 11-7082-treated group, unpaired two-tailed t-test.m Immunofluorescence images of intestinal villi from APC Min -Villin-RasV12 mice administered with PBS or SN50.Magnified images depict basal (an arrow) or apical extrusion of transformed cells from PBS-or SN50-administered mice, respectively.Scale bars, 10 μm (c, f, h-k, m).n Quantification of extrusion of APC Min /RasV12-transformed cells in the absence of the presence of SN50.Data are mean ± s.e.m. n = three independent experiments.*P = 0.0486 for basally extruded cells between PBS-and SN50administrated group, unpaired two-tailed t-test.